Acidovorax spp., Pseudomonas spp., Novosphingobium spp. and Sphingomonas spp., were the dominant members of the bacterial community (about 71%) at the beginning of the biodegradation; many of these organisms have previously been shown to biodegrade BDE-209 and other polybrominated diphenyl ether (PBDE) congeners. The Achromobacter sp. that were isolated  were especially effective during the BDE-209 degradation. These results indicated the effectiveness of the sequential UV photolysis and biodegradation for treating certain BDE-209-contaminated solids; e.g., clays; in bioreactors containing such solids as aqueous slurries. Achieving a similar treatment effectiveness for more heterogeneous solids containing natural organic matter, e.g., surface solids, appears to be significantly more difficult. Further investigations are needed in order to understand the great difference between the clay-water or soil-water slurries.Decabrominated diphenyl ether (BDE-209) is a primary component of the brominated flame retardants used in a variety of industrial and domestic applications. BDE-209 bioaccumulates in aquatic organisms and has been identified as an emerging contaminant that threatens human and ecosystem health. Sequential photolysis-microbial biodegradation processes were utilized here to treat BDE-209 in clay- or soil-water slurries. The removal efficiency of BDE-209 in the clay-water slurries was high; i.e., 96.5%, while that in the soil-water slurries was minimal. In the clay-water slurries the first order rate constants for the UV photolysis and biodegradation of BDE-209 were 0.017 1/day and 0.026 1/day, respectively. UV wavelength and intensity strongly influenced the BDE-209 photolysis and the subsequent biodegradation of photolytic products. Facultative chemotrophic bacteria, including  Large quantities of solid waste containing BDE-209, in particular electronic or electrical wastes (e-waste), continues to be discarded inappropriately resulting in the release of BDE-209 into the environment [Polybrominated diphenyl ethers (PBDEs), especially the penta-, octa- and deca- brominated congeners, constitute a major class of the commercial brominated flame retardants (BFR). These BFRs are used in plastics, textiles and in wire insulation, and hence are found in many commercial products such as automobiles. Decabrominated diphenyl ether BDE-209) has continued to be used as a BFR even after the global prohibition of penta-BDE and octa-BDE, although its use has been restricted in the EU and the USA under the United Nations Stockholm Convention (ironment . As a re09 has coPBDEs have been identified as emerging contaminants (EC), which are defined as chemicals that have been detected in the environment that may have ecological or human health impacts but are not sufficiently regulated under current environmental law. Many PBDEs congeners, such as tetra-BDEs to hepta-BDEs, have been described as both persistent organic pollutants (POPs) and endocrine disrupting chemicals (EDCs). Environmental scientists have begun to pay attention to the chronic toxicity of these chemicals because they typically have long half-lives in the environment. The human health effects of BDE-209 have been shown to involve the liver, heart, kidneys and the thyroid, and to include various reproductive, developmental and neurobehavioral effects. For example, using Sprague-Dawley rats as a mammalian model, BDE-209 has been shown to induce oxidative stress and inflammation, leading to endothelial dysfunction and morphological/ultrastructural damage to the heart and the abdominal aorta . Human e2 [BDE-209 photodegradation occurs when free-radicals are formed by UVA and UVB radiation which then attacks the bromines present in PBDEs. UV photolysis to remove PBDEs has been applied to liquid matrices including organic solvents and water, and to gas and solid phases including TiO2 . UV phot2 . The UV 2 ,7. TheseA sequential treatment process involving both UV radiation and biodegradation has demonstrated potential as a technique for POP/EDC removal. Prior studies have shown a variety of UV-biological treatment processes that have been used to remove target POPs/EDCs , chlorphenols and pesticides) in various aqueous solutions and organic solvents of differing compositions . This apad-situ or ex-situ remediation technologies [Our aim in this study was to evaluate a sequential photolysis-bioremediation process for the treatment of BDE-209-contaminated natural solids in an aqueous slurry system. Such a system avoided the need to dewater, and allowed for the optimal mixing and control of redox conditions. Importantly, this type of system is ubiquitous in aquatic ecosystems  and agricultural farms , which are common sinks for the accumulation of POPs/EDCs ,2. Moreonologies . Treatmenologies . BecauseThe BDE-209  used in this study had a purity above 99%. A standard solution of BDE-209  was made by dissolving BDE-209 in hexane and this was used when required for the analysis of the concentration of BDE-209. All organic solvents were HPLC grade and had a purity of greater than 99.9%. All the other chemicals used in this study were reagent grade and had a purity of greater than 99%. The Milli-Q water was double-distilled and deionized by a Millipore water purification system.A bacterial mixed culture capable of BDE-209 biodegradation was previously obtained by sampling activate sludge that contained 256.8 BDE-209 ng/g; this was obtained from a biological treatment plant at Nei-Hu Wastewater Treatment Plant . The details of the enrichment process used on the Nei-Hu bacterial mixed culture was outlined in our previous research .Two types of sorbents were selected to be used as a sorbent/water system. The physical-chemical characteristics of these sorbents are shown in 3 and 827 cm3, respectively. A polypropylene plastic board was used to cover the top of the bioreactor; this meant that there was no exposure to UV irradiation from the external environment, only from the UV lamp inside. The UV lamps used in this study were a 365-nm UVA lamp  and a 312-nm UVB lamp ; these were setup as alternatives in the middle of the quartz inner tank. UV light is able to easily penetrate the quartz into the sorbent/water system and this radiation is able to generate significant free radicals; these will then be able to chemically alter then cleave the structure of BDE-209 and its metabolites. The intensity of UV irradiation on the outer surface of the inner quartz tank was measured and was found to range from 1.64 to 2.71 mw/cm2 when the 365-nm UVA lamp was used and from 0.58 to 0.97 mw/cm2 when the 312 nm UVB lamp was used. The sorbent/water systems, namely the clay/water slurry system and the soil/water slurry system, were added individually to the space between the two tanks. The complete mixing of both sorbent/water systems during the BDE-209 photolysis/biodegradation was brought about by the use of a magnetic stir plate set at 300 rpm. Since the heat from the UV irradiation system was continuously entering the SSR, cooling via a circulating system was set up in order to keep the temperature of the system at a steady 25 °C during all the experiments.The detailed design of the SSR that was used for the sequential treatment of BDE-209 by the UV photolysis and biodegradation is shown in 2) was set up in order to avoid a reductive debromination by outside radiation or by various other chemical reactions. The samples were collected on the first, seventh, 14th, 21st, 28th and 35th days. A constant sorbent-to-water ratio was maintained in the SSR in order to prevent the sampling procedure from having an effect on the BDE-209 degradation. After the UV photolysis, the BDE-209 biodegradation was carried out in the same bioreactor system and this involved a number of steps. Specifically, over a total of eight days (from the 35th day to the 42nd day), the UV-pretreated sorbent/water samples were removed from the photolysis process and set up as a biodegradation process using the same bioreactor. The change consisted of three major processes. Firstly, the aqueous solution present in the sorbent/water bioreactor was discarded. Secondly, the sorbents were autoclaved at least twice to remove any bacteria that had grown in the reactor during the UV photolysis. Thirdly, a mineral salt basal (MSB) medium [5 CFU/mL of mixed bacterial culture when grown on R2A agar [2); the experiment continued from day 43 to day 140 (98 days). The sorbent/water system was sampled regularly on the 43rd, 74th, 105th and 140th day to allow for analysis. A constant sorbent-to-water ratio was maintained in order to prevent the sampling procedure from affecting the BDE-209 biodegradation. The control sorbent/water systems were used, the first stage of which consisted of biodegradation alone in an absolutely dark environment (no UV irradiation), while the second stage consisted of sterilized BDE-209-MSB that had been poisoned with 32.5g HgCl2. These were analyzed in a similar manner to that of the non-control systems.The designed sequential treatment process for the BDE-209 degradation using the two different sorbent/water systems was carried out in the SSR described above. The first treatment process involved UV photolysis to degrade BDE-209 in a sorbent/water system for 35 days. An absolutely dark environment (0 mw/cmv/v). The solid sample was extracted twice for 30 min using an ultrasonic water bath (output energy of 200 W at a frequency of 40 kH) at room temperature. The water level in the bath was adjusted to be equal to that of the extraction solvent in the PTFE centrifuge tube. For the GC/MS analysis, purer extracts were required and an extra cleaning process was introduced that involved a 16 × 300 mm glass chromatography column packed with self-prepared 60–200 mesh acidic silica gel and specific glass wool. The sample extract was eluted from the acidic silica gel column using 20 mL hexane and then concentrated to near dryness by rotary evaporation. Finally, the residue was dissolved in 1.0 mL hexane in a flask and filtered through a 0.22 μm PTFE syringe filter. A final volume of 1 μL was injected during the chromatographic analysis. The stock solutions of BDE-209 were prepared by serial dilution in hexane and these were stored until use in an individual dark-brown glass container at 4 °C to prevent BDE-209 photolysis.An ultrasound extraction method was selected as an appropriate approach to be used for the analysis of BDE-209. For the GC analysis, a solid sample was obtained from the sorbent/water system by high-speed centrifugation in a polytetrafluoroethylene centrifuge tube. Precisely 1.000 g of the solid sample was then extracted using 10.0 mL of solvent, namely a mixture of hexane and acetone  helium (99.999% purity) and 5% (v/v) refined methane at a high purity and the carrier gas ran at a constant flow rate of 10 mL/min. The injection port and detector of the GC/PDECD were maintained at 320 °C and 380 °C, respectively. The temperature of the GC oven was initially set at 110 °C for 1 min and programmed so that the temperature increased at 40 °C/min until 200 °C was reached at which point it was held at 200 °C for 3 min; this was followed by a final ramp up to 330 °C at a rate of 10 °C/min. The temperature was then maintained at 330 °C for 4.75 min. The voltage of the PDECD was set at 30 V. Finally, 3 μL of the sample extract dissolved in hexane or 3 μL of the standard BDE-209 solution dissolved in hexane was injected into the GC/PDECD in the splitless mode. The method detection limit (MDL) for measuring BDE-209 in the sorbent/water systems was found to be 0.032 μg/g.The concentration of BDE-209 was measured by GC (HP 5890 Series II) using a pulsed discharge electron capture detector . The conditions used for the BDE-209 analysis were as follows: a Restek® DB-5HT capillary column was used for the separation of the PBDE congeners. The carrier gas was helium with a purity of 99.999% and was delivered at a rate of 1 mL/min. The GC oven temperature was programmed to increase in temperature from 110 °C (5 m) to 200 °C (5.5 m) at 40 °C 1/min then to increase to 330 °C at 10 °C 1/min; this temperature was then held for 2.25 m. The injector temperature was set at 320 °C and the separation was carried out in the splitless mode. The sample consisted of 1 mL of either the sample extract or the standard solution, both in hexane. To condition the MS, electron ionization (EI) was performed at 38 eV. The electron multiplier voltage and emission current were set up at 10 kV and 500 mA, respectively. The source temperature of the MS was set at 320 °C. The resolving power of the analyzer was >10,000. The MS was operated in the selected ion monitoring (SIM) mode and used eight descriptors to analyze the 23 PBDE congeners.The 23 PBDE congeners were analyzed using a GC (HP 6890) equipped with an GC PAL autosampler  that was coupled to an HSMS-700 high-performance double focusing magnetic sector mass spectrometer . An Agilenthttp://basespace.illumina.com/. The sequences found on the day 43, day 105 and day 140 samples, which were generated by the Illumina 454 sequencing platform, are shown in Metagenome analysis has become one of the most powerful techniques available for the analysis of the bacterial communities present in different ecosystems. Illumina sequencing technology is a widely adopted next-generation sequencing (NGS) technology that has been responsible for generating more than 90% of the world’s sequencing data. This approach was applied to assess the changes in the bacterial communities during the BDE-209 biodegradation at different time points . DNA samples were extracted using a Soil Genomic DNA Purification Kit . The bacterial communities were analyzed using the 27F-519R primer set to amplify the V1–V3 domains of 16S rDNA on an Illumina 454 stage  . The DNAThe bacterial strains capable of using BDE-209 as a sole carbon source were isolated during the BDE-209 biodegradation in the sorbent/water system. To do this, 10 mL the sorbent/water mixture was sampled and diluted 10-times serially on days 43, 74, 105 and 140. A 0.2 μL inoculum was spread on the purified agar plates that contained 20 μg/L  BDE-209 as the sole carbon source plus MSB. To prevent bacterial strains from utilizing the organic residues present in agar as a carbon source, purified agar was obtained by washing the agar powder by Milli-Q water at least three times before medium preparation. Pure strains as single colonies were identified after incubation at 25 °C for 14 days. These isolated strains were confirmed as being able to grow well on the BDE-209-MSB agar plates by the streak method technique and this streaking was repeated at least 3–5 times. Finally, these strains were identified by the Mission Biotech company (Taiwan) based on the V1–V8 domain sequences of their 16s rDNA regions.A Biolog MT2 microplate -based assay was selected for the rapid identification and evaluation of the ability of a given bacterial strain to utilize BDE-209 as a sole carbon substrate. Each Biolog MT2 microplate well was created as a sole carbon source substrate well (20 mg/L BDE-209) in MSB; in addition, an equal concentration of tetrazolium violet dye, which is sensitive to the oxidation of a carbon source and bacterial respiration, was added . The mic2r = 0.92) and 33 days, respectively. Considering these results, the BDE-209 biodegradation following UVB photolysis using the clay/water slurry system was selected as the model multi-stage system. Using this system, the BDE-209 biodegradation commenced at concentration of 7.72 ± 2.01 μg/g following inoculation on the 43rd day, and continued to a final concentration of 0.60 ± 0.12 μg/g on the 140th day. The extent of the BDE-209 biodegradation was c. 92.23%, and the first-order rate constant and half-life of the BDE-209 biodegradation were c. 0.026 1/day (2r = 0.99) and 22 days, respectively. Thus, the total BDE-209 removed by the sequential UVB photolysis-biodegradation was c. 96.47% in the clay/water slurry system over a period of 140 days. In the sterile (HgCl2) control, the concentration of BDE-209 remained in the range of 16.0 ± 0.2 μg/g to 19.4 ± 1.6 μg/g during the 140-day treatment period.The individual and combined effectiveness of the sequential process involving UV photolysis and biodegradation for the treatment of BDE-209 in the bioreactor containing clay- and soil-water slurries are shown in Acidovorax spp. (58.88%), Sediminibacterium spp. (8.67%), Novosphingobium spp. (6.56%), Pseudomonas spp. (5.12%), Janthinobacterium spp. (3.02%), Curvibacter spp. (2.67%) and Bradyrhizobium spp. (1.48%). On day 105, the major species (>3%) present consisted of Alicycliphilus spp. (20.37%), Acidovorax spp. (17.89%), Pseudomonas spp. (12.07%), Ramlibacter spp. (7.13%), Novosphingobium spp. (6.72%), Thauera spp. (3.60%) and Sphingomonas spp. (3.04%). Finally, on day 140, the eight major species detected were Desulfomonile spp. (43.21%), Sphingomonas spp. (7.00%), Pseudomonas spp. (6.08%), Novosphingobium spp. (4.73%), Symbiobacterium spp. (2.98%), Acidovorax spp. (2.07%) and Sediminibacterium spp. (1.96%).The phyla Proteobacteria and Bacteroidetes appeared to play important roles in the biodegradation of BDE-209 in the clay/water slurry system accounting for 82.13% to 90.47% and 2.31% to 8.78% of the total bacterial population, respectively. The changes in the bacterial community that occurred during BDE-209 biodegradation based on a class level analysis are shown in Acinetobacter sp. strains which are members of the γ-proteobacteria , three Achromobacter sp. strains which are members of the β-proteobacteria , one Pseudomonas sp. strain which is a γ-proteobacteria (NH-5) and one Bacillus sp. strain which is a member of the Firmicutes (NH-3). Using the Biolog MT2 microplates method, Achromobacter sp. NH-2 was characterized as being able to strongly utilize BDE-209 as a sole carbon source; the comparative abilities to utilize BDE-209 as a sole carbon source were medium in four of the above mentioned strains  and weak in the three additional strains . Four bacterial strains  were isolated on day 43 after the pretreatment process involving the photolysis acted as an enhancing factor in terms of the biodegradation process. These strains were able to utilize the metabolites created by the photolytic process, namely the low bromides PBDEs and used these for biological co-metabolism with BDE-209 in the SSR.The bacterial strains isolated from the samples taken on days 43, 105 and 140 during the biodegradation in the clay/water slurry reactor and their ability to utilize the BDE-209 as a sole carbon source are shown in k = 0.017 1/day during 35 days of treatment. In contrast, the UVB-based photolysis process was unable to successfully degrade BDE-209 in the soil/water slurry system (k = 0 1/day). A prior study showed similar differences in the degradation of BDE-209 associated with clays vs. soils. Specifically, in a slurry reactor with continuous UV irradiation (300–400 nm) for 14 days the results were as follows: Na+-montmorillonite  (k = 0.0192 1/day) > kaolinite  (k = 0.0158 1/day) > loam sediment with 16.4% organic carbon (k = 0.0046 1/day) [k = 0.017 1/day) by UVB irradiation in the clay-water slurry was slower than that in the Na+-montmorillonite, which was likely due to the differences in the experimental setup and conditions, such as the process in a saturated water environment or the 312nm-UVB irradiation in the Ca-montmorillonite/water system. Another study found that the percentage of BDE-209 remaining after the treatment of various geosorbents with UV irradiation (300–400 nm) for 64 h were: lake sediment (57%) > sand (21%) > silica gel (1%), and in two samples the significant levels of BDE-209 remained after 244 h, i.e., a Nordic reference soil (38%) and the lake sediment (26%) [UVB irradiation was found to be an effective process for degrading BDE-209 in the clay/water slurry system yielding a first order rate constant 6 1/day) . Na+-mon) k = 0.08 1/day >The two different sorbents used (clay vs. soil) have been previously shown to affect the rate of BDE-209 biodegradation. In the clay/water slurries, the BDE-209 biodegradation displayed first order kinetics with a rate constant of 0.026 1/day , almost A primary mechanism of the PBDE photodegradation includes the consecutive reductive debromination and intramolecular elimination of HBr in soils . The pos® 2120 UV plus, Mecasys) was identified at 312 nm or 365 nm UV irradiation. The absorption spectrum of BDE-209 itself and in clay/water slurry systems are shown in ad-situ or ex-situ remediation. In addition, UVC light with a shorter-wave (280–200 nm) in practice damages human DNA and creates an increased risk of skin cancer. Indigenous microorganisms in the soil are unable to survive during UVC irradiation. This study selected UVB irradiation to reduce the health risks to staff associated with UVC irradiation exposure during the SSR operation.The photolysis rate of BDE-209 is influenced by the wavelength and intensity of UV irradiation, which can affect the quantum yield ,17. UV wTwo other environmental factors pertinent to the reactor system used here might provide electrons in addition to those produced by the UVB irradiation itself. Firstly, clay minerals are known to have a significant electron-donating ability during debromination . The mon−8 Einstein/L.s for 3–6 h) [Pretreatment by 35-day UV irradiation appeared to influence the BDE-209 biodegradation in the clay/water slurry system used herein. This UV photolysis pretreatment appeared to increase the rate of BDE-209 biodegradation in the clay/water slurry system by allowing co-metabolism during the subsequent biodegradation step when compared to systems without prior UV treatment. The first-order-rate constant from this study (with UV pretreatment) was 0.026 1/day, which is almost 2.8-fold higher (0.0092 1/day) than that obtained for biodegradation under the same incubation conditions and using the same bacterial inoculation (obtained from Nei-Hu WWTP) but lacking UV pretreatment [r 3–6 h) .Achromobacter spp., Acinetobacter spp. and a Bacillus sp., which were able to utilize BDE-209 as a sole carbon source. Achromobacter are Gram-negative β-proteobacteria. They are strictly aerobic and commonly found in water and soils. One example, Achromobacter piechaudii, isolated from contaminated desert soil, was able to biodegrade 2,4,6-tribromophenol and produce bromide under aerobic conditions [Achrormobacter can use specific polychlorinated biphenyls (PCB) congeners as a sole carbon and energy source for growth; these chemicals have structural similarity to PBDE congeners [Acinetobacter strains  were also isolated; these are γ-proteobacteria and were confirmed to biodegrade PBDEs. Acinetobacter have been reported to grow on agar containing 0.5 mg/L BDE-209 and hence are considered as BDE-209-degrading bacteria; these strains were isolated from river sediments [Acinetobacter sp. able to carry out BDE-100 biodegradation [Acinetobacter spp. present in a mixed culture  increased when alternative electron donors such as methanol and ethanol were provided [Bacillus sp. strain capable of BDE-209 biodegradation was identified. A previous study showed that the metal-resistant bacterial strain Bacillus cereus JP12 was able to use BDE-209 as a sole carbon and energy source when grown on MSB. Using this strain, more than 88% of the BDE-209 present (1 mg/L) was biodegraded over 12 days at pH 6.0 and 30 °C [Pseudomonas with a medium ability to utilize BDE-209 was isolated during our study on day 105.Eight bacterial strains capable of degrading PBDEs or their biodegradation products were isolated from the clay/water slurry system. These strains included nditions . In addiongeners . Three Aediments . A mixedradation . In anotprovided . In the nd 30 °C . A singlAcidovorax spp., which is a member of the β-proteobacteria, present in the total bacterial population, was found to be the highest at 58.88% on day 43 in our clay/water slurry system; this was calculated as a relative abundance against the total community. Acidovorax is Gram-negative and a member of the β-proteobacteria; this species is known to be able to degrade many POPs/EDCs, especially aromatic compounds in oligotrophic groundwater [Acidovorax spp. have been identified in the high-level BDE-209-contaminated microcosm containing lower levels of SOM soil [Acidovorax spp. in the lower-SOM clay/water system was able to utilize BDE-209 (7.72 ± 2.01 μg/L) as a single carbon source by generating target enzymes such as those encoded by the Phn genes and Bph genes, both of which are capable of cleaving the structure of diphenyl ether [Acidovorax spp. can therefore be seen to decrease to 17.89% on day 105 and 2.09% on day 140 during biodegradation. On the other hand, Sediminibacterium spp., present in the original bacterial mixed culture inoculum was dominant during the second stage of BDE-209 biodegradation and was then replaced by other bacterial species. The percentage of Sediminibacterium spp. present during BDE-209 biodegradation in the samples collected from the clay/water slurries gradually decreased from 8.67% on day 43, to 2.32% on day 104 and then to 1.96% on day 140; this was calculated as a relative abundance against the total community. Sediminibacterium spp. are aerobic bacteria often found in aquatic sediment, activated sludge, surface water and waste reservoirs under solar irradiation. A relatively high percentage (11.76%) of S. ginsengisoli was identified in the microcosms under continuous exposure to 365-nm UV irradiation at 1.5~2.0 mw/cm2 in the presence of a high level of BDE-209, compared to their absence in the dark environment [Many bacteria identified in this study were capable of biodegrading BDE-209 as well as other PBDE congeners and aromatic POPs. For example, the percentage of undwater . The SOMSOM soil . In thisyl ether ,33. Whenironment .Alicycliphilus spp. detected during BDE-209 biodegradation was 20.37% on day 105, which should be compared to 0.52% on day 43 and 1.13% on day 140; this was calculated as a relative abundance against the total community. Alicycliphilus spp. are facultative Gram-negative bacteria commonly found in activated sludge from WWTP, waterbodies and sediments, and are known to degrade a wide variety of aromatic compounds. Alicycliphilus spp. have been found to degrade pentachlorophenol in aerobic granular sludge and they have also been shown to be responsible for the breakdown of a phenol during the activated-sludge process [A. denitrificans K601 has been reported to be able to biodegrade BDE-209 [Pseudomonas spp. are facultative Gram-negative bacteria found during the course of BDE-209 biodegradation in all the collected samples; they made up 5.12% of the total bacterial population on day 43, were dominant at 12.07% on day 105 and were still quite dominant at 6.08% on day 140, calculated as a relative abundance against the total community. Pseudomonas spp. have been shown to metabolize benzene and other aromatic compounds [P. aeruginosa, when added to PBDE-contaminated soil, was found to biodegrade BDE-209 [P. dioxanivorans CB1190 have also been shown to biodegrade lower brominated PBDEs [Novosphingobium spp. present were α-Proteobacteria Gram-negative aerobic bacteria which are commonly found in waterbodies, soil and sediment. The percentages of Novosphingobium spp. present during BDE-209 biodegradation in the clay/water slurries were 6.56% on day 43, a dominant 6.72% on day 105 and 4.73% on day 140; this was calculated as a relative abundance against the total community. Novosphingobium spp. have been shown to degrade many POPs/EDCs including chlorophenols and PAHs [N. chloroacetimidivorans isolated from activated sludge have been shown to biodegrade pesticides including butachlor, acetochlor and alachlor [Ramibacter spp. detected during BDE-209 biodegradation reached 7.13% on day 105. Ramibacter spp. are β-proteobacteria Gram-negative aerobic bacteria that have been found in desert sands. R. tataouinensis has been reported to possess significant DNA repair mechanisms in the form of highly active superoxide dismutase that is able to remove reactive oxygen species allowing the bacterium to tolerate oxidative stress. It is inferred that Ramibacter spp. might be irrelevant to the biodegradation of BDE-209 and its metabolites, but these bacteria may be able to survive well in the clay/water slurries when there is continuous UV irradiation.Most of the bacteria identified as present in the clay/water slurries bacterial community were dominant on day 105, indicating that they played an important role in BDE-209 degradation and possibly the biodegradation of other metabolites. For example, the highest percentage of  process . A. deni BDE-209 . Moreoveompounds . P. aeru BDE-209 . BDE-209ed PBDEs . In addiand PAHs ,40. N. calachlor . FurtherDesulfomonile spp. was 43.21% on day 140 of BDE-209 biodegradation. Desulfomonile spp. are Gram-negative strictly anaerobic bacteria often found in activated sludge from WWTP and in river sediments. This dominant species is likely to be present in a range of different anaerobic micro-environments within the clay/water slurry system where there are higher levels of electron acceptors such as PBDEs. Chlorophenol and trichlorophenol are reductively dechlorinated by Desulfomonile tiedjei DCB-1 [Sphingomonas spp. were identified on day 105 (3.04%) and day 140 (dominant at 7.00%) in the clay/water slurry system; this was calculated as a relative abundance against the total community, possibly because they were unitizing the low-brominated PBDEs that were present. Sphingomonas sp. are Gram-negative facultative anaerobes that have been shown to have the ability to biodegrade low-brominated PBDEs. Sphingomonas sp. SS3 has been shown to biodegrade 4-bromodiphenyl ether [Sphingomonas sp. PH-07 is capable of biodegrading PBDE congeners such as 4-Bromodiphenyl ether, 2,4-dibromodiphenyl ether and BDE-15 when they are present in a diphenyl ether solution [The percentage of ei DCB-1 . It can yl ether . SphingoAcidovorax spp. and Sediminibacterium spp. were dominant during the initial period (day 43) during which there was the biodegradation of the high-brominated PBDEs . Alicycliphilus spp., Acidovorax spp., and Pseudomonas spp. were dominant during the middle period (day 105), which involved the utilization of various metabolites such as the low-brominated PBDEs and hydroxylated PBDEs (OH-PBDEs). Desulfomonile spp. were dominant during the last period (day 140), which suggested that biological debromination was occurring in clay-water slurry microenvironments. Various bacterial strains such as Achromobacter spp., that were isolated from the sequential UV photolysis-microbial degradation process, were found to have the ability to utilize BDE-209 as sole carbon source. These findings provide the basis of a novel approach for degrading BDE-209 and similar ECs. More detailed experiments are required to further optimize the design and functioning of a practical ad-situ or ex-situ system for the remediation of POP/EDC-contaminated solids using a slurry bioreactor.A sequential UV photolysis-microbial degradation process was developed and characterized which effectively removed BDE-209 in a clay/water slurry system. The pure clay mineral used (montmorillonite), along with the water molecules present, are likely to have provided sufficient electrons for a series of photolytic reactions. The dominant bacterial communities identified have been shown in prior studies to have the capacity for PBDE biodegradation. The relative abundances of major genera at the community level were found to have changed significantly at the different stages of biodegradation.


There are few effective therapies for coronavirus disease 2019 (COVID-19) upon the outbreak of the pandemic. To compare the effectiveness of a novel genetically engineered recombinant super-compound interferon (rSIFN-co) with traditional interferon-alpha added to baseline antiviral agents (lopinavir–ritonavir or umifenovir) for the treatment of moderate-to-severe COVID-19.In this multicenter randomized (1:1) trial, patients hospitalized with moderate-to-severe COVID-19 received either rSIFN-co nebulization or interferon-alpha nebulization added to baseline antiviral agents for no more than 28 days. The primary endpoint was the time to clinical improvement. Secondary endpoints included the overall rate of clinical improvement assessed on day 28, the time to radiological improvement and virus nucleic acid negative conversion.p = .019); the overall rate of clinical improvement on day 28 was 93.5% versus 77.1% ; the time to radiological improvement was 8.0 days versus 10.0 days (p = .002), the time to virus nucleic acid negative conversion was 7.0 days versus 10.0 days (p = .018) in the rSIFN-co and interferon alpha arms, respectively. Adverse events were balanced with no deaths among groups.A total of 94 patients were included in the safety set . The time to clinical improvement was 11.5 days versus 14.0 days  upon the outbreak of the pandemic. Interferon alphas, by inducing both innate and adaptive immune responses, have shown clinical efficacy in treating severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus.In this multicenter, head-to-head, randomized, clinical trial which included 94 participants with moderate-to-severe COVID-19, the rSIFN-co plus antiviral agents (lopinavir–ritonavir or umifenovir) was associated with a shorter time of clinical improvement than interferon-alpha plus antiviral agents.rSIFN-co was associated with a shorter time of clinical improvement than traditional interferon-alpha in the treatment of moderate-to-severe COVID-19 when combined with baseline antiviral agents. rSIFN-co therapy alone or combined with other antiviral therapy is worth to be further studied. The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected more than fifty-two million people worldwide as of November 13, 2020 . Thus faDiagnosis and Treatment Protocol for Novel Coronavirus Pneumonia released by the National Health Commission of China  39.8 to 63.3), and 46.8% of the patients were male. In the rSIFN-co group there were 39 moderate cases and 7 severe cases, while in the interferon-alpha group, there were 43 moderate cases and 5 severe cases. The median interval between symptom onset and randomization was 14.0 days (IQR 5.0 to 30.0) in the rSIFN-co group and 14.5 days (IQR 7.0 to 31.0) in the interferon-alpha group. Some patients  were transferred to other hospitals according to the government’s unified deployment. The detailed information about the status of these patients when transferred are summarized in Supplementary Table 3. The outcomes of these 11 patients were assessed when transferred and included in the final analysis.One patient in the interferon-alpha group suffered disease deterioration and was transferred to another hospital, which was designated for critical ill patients. The other 10 patients   .p = .002) (p = .018)  . The tim = .002) . The tim = .018) . The ove = .018) . The ove = .018) . One patSupplementary Table 4), most of which were classified as grade 1 or 2 with decreased appetite being the most common in both groups. There were no SAEs reported in the rSIFN-co group, while one patient had a secondary bacterial infection followed by respiratory failure in the interferon-alpha group. The latter case was deemed to be a non-treatment related SAE, and interferon-alpha administration was ceased and invasive mechanical ventilation was applied to this patient. There were no deaths in either group between the initiation of medication and day 28.AEs were reported by 13 (28.3%) of the 46 patients in the rSIFN-co group and 18 (38.5%) of the 48 patients in the interferon-alpha group  and no patient was given invasive mechanical ventilation or extracorporeal membrane oxygenation at the time of enrolment. During the study period, patients with COVID-19 were admitted to hospitals of different levels according to the severity of the disease following the guidance of health administration department. Our patients were recruited from five hospitals designated for moderate-to-severe patients. The time interval between symptom onset and randomization varied among patients, and some of the participants had received symptomatic treatment and/or supportive care, but without clinical improvement. We distributed the participants evenly between the two groups by randomization. Although some patients were transferred during the middle of the study and failed to complete the whole treatment regimen according to the government’s unified deployment, all of them were evaluated before transfer and were included in the final analysis.Interferon-alphas alone or combined with other antiviral agents have antiviral effects on multiple types of viral infections, such as SARS-CoV and MERS-CoV ,12,20–23However, severe COVID-19 patients often develop acute respiratory distress syndrome (ARDS) or secondary haemophagocytic lymphohistiocytosis (sHLH) ,27. BothFurthermore, studies showed type I interferon deficiency could be a hallmark of COVID-19 and at least 10% of patients with COVID-19 pneumonia have neutralizing auto-antibodies against type I interferons, which highlights the crucial role of type I interferons in protective immunity against SARS-CoV-2 ,37. A raPrevious studies on interferon-alpha showed that a few patients had influenza-like symptoms, such as pyrexia, myalgia, and rigours, after receiving treatment . The preOur study has several limitations. Firstly, the total number of trial patients was small, although it is not uncommon for an exploratory study, further studies are encouraged to confirm these results with more patients. Secondly, the median interval between symptom onset and randomization was longer in our study than that in other reports and some patients received symptomatic treatment (such as cough relief and fever reduction) and/or supportive care before randomization ,3. ThirdIn conclusion, rSIFN-co was associated with a shorter time of clinical improvement than traditional interferon-alpha in the treatment of moderate-to-severe COVID-19 when combined with baseline antiviral agents. Larger studies with rSIFN-co therapy alone or combined with other antiviral therapy that include a broader range of patients with COVID-19 are worth to be conducted.Click here for additional data file.


No significant differences were observed in the proportion of central memory and effector memory T cells or early activated CD25+ cells between any of the test groups. Our results suggest that EE, irrespective of duration, enhances the numbers of microglia, but long-term EE is required to modify astrocyte number and peripheral T cell proportions in middle-aged mice. Our findings provide new insights into the therapeutic effects of EE on various brain disorders, which may be at least partly mediated by glial and neuroimmune modulation.Environmental enrichment (EE) has been shown to modulate behavior and immunity. We recently reported that both short and long-term EE enhance baseline locomotion and alleviate depressive-like behavior, but only long-term EE affects locomotion adversely in a threatening environment and enhances anxiety-like behavior in middle-age mice. We have now investigated whether the observed changes in behavior after short- and long-term EE were associated with underlying immune changes. Hence, at the end of behavioral testing, mice were sacrificed, and brains and cervical lymph nodes were collected to investigate the differential effects of the duration of EE (short- and long-term) on the number of immunopositive glial cells in the dentate gyrus, CA1, CA2, and CA3 regions of the hippocampus and proportions of T cell subsets in the cervical lymph nodes using immunohistochemistry and flow cytometry, respectively. EE, regardless of duration, caused an increase in microglia number within the dentate gyrus, CA1 and CA3 hippocampal regions, but only long-term EE increased astrocytes number within the dentate gyrus and CA3 hippocampal regions. A significantly higher proportion of CD8 Environmental enrichment in rodents has been studied extensively in the last decade for its beneficial effects on neuroimmune mechanisms that enhanced our understanding and approach in dealing with psychiatric disorders. EE promotes cognitive stimuli in the experimental animals with the use of running wheels, novel objects, puzzles  and accessories  and microglia (IBA1+) antigen expression within the dentate gyrus, but not in the CA1, CA3, or cortex of adult male rats that were housed for 12 h in an enriched environment  in the modulation of neuroimmune mechanisms remains unclear. In our recently published work, we reported an increase in baseline locomotion and decrease in depressive-like behavior after both short and long-term EE in middle-aged mice. Furthermore, long-term EE affected locomotion adversely in a threatening environment and was found to be anxiogenic , parental substrain Nhsd , were bred in-house in the laboratory animal services (LAS) facility at the University of Adelaide and housed in individually ventilated cages (IVCs) under controlled conditions of temperature (21 ± 1°C), humidity (55%) and a 12–12 h dark-light cycle. During the experimental timeline, the C57BL/6Nhsd mouse line was inbred between 9 and 13 generations. Mice were provided ad libitum standard laboratory food and water. C57BL/6 mice were selected for the study since they have been characterized well in terms of humoral and cellular neuroimmune responses to environmental factors , and all guidelines as prescribed for handling the experimental animals were followed during the study.n = 12–16). Mice were randomly paired  and transferred to open top cages (2 mice per cage). Control mice received no treatment and were kept in cages with the following dimensions: 48.5 × 15.5 × 12 cm. EE mice were kept in plexiglass cages with dimensions: 37 × 20.5 × 13.5 cm, as these had more breadth and depth to provide extra space for the objects associated with EE.Once the desired age (12 weeks or 8 months of age) was reached, those mice without signs of injury or illness, hence not challenged immunologically, were randomly allocated into either treatment (short-term and long-term EE) or control groups  and extra bedding as per previously published protocols  was applied to the slides which were then left to incubate overnight, allowing primary antibodies to bind to the target antigen. On day 2, the IgG biotinylated antibody of rabbit (same as primary antibodies) was added and allowed to react with primary antibodies for 30 min. The formed immune complex was then further amplified by incubating slides with a biotin-binding protein, streptavidin-peroxidase conjugate, for 60 min. The immune complex was then visualized with precipitation of DAB in the presence of hydrogen peroxide. Slides were washed to remove excess DAB and lightly counterstained with hematoxylin, dehydrated and mounted with DePex.All slides were digitally scanned  and then viewed with the associated software NDP view (version 1.2.2.5). Immunopositive cells in the dentate gyrus, CA1, CA2, and CA3 regions of the hippocampus were counted manually for statistical analysis. The hippocampus, in particular, the dentate gyrus region, has been shown to play a vital role in the regulation of behavior and memory in response to external stimulus and shows alterations after EE  to get the number of cells/mm2. The average of six sections represented the value for one mouse and was utilized during statistical analysis. It is important to note that unlike the dentate gyrus, the other regions of the hippocampus are not clearly demarcated. Hence, the sampling boxes technique was utilized to analyze the number of immunopositive glial cells in the CA1, CA2, and CA3 regions of the hippocampus. To achieve this objective, square boxes of equal area were drawn in the CA1, CA2, and CA3 regions of the hippocampus, and the cells were counted within boxes. For each section, the cell count was then divided with the area of the box (in mm2) to get the number of cells/mm2. The average of six sections represented the value for one mouse and was utilized during statistical analysis.Freehand boxes were drawn to cover the entire dentate gyrus regions of the six stained sections followed by counting of the cells within the boxes. For each section, the count of cells was then divided with the area of the box , and early activated T cell phenotype (CD25+).FACS was applied for the detection of T cell proportions and characterization of their phenotype in the cervical lymph nodes of mice. This included CD46 cells/ml. 250 μL of the cell suspension was then washed once with FACS buffer  and blocked with 10 μL 0.5 mg/mL Fc block. An eight-color staining panel was used to characterize the CD4+ and CD8+ T cells. Unstained cells were used to gate out autofluorescent cells while single stained and fluorescence minus one (FMO) stained cells were used to control for spectral overlap or distinguishing between negative and positive cells, respectively (non-specific binding). Cells were incubated for 30 min at room temperature with the respective mAbs .Cervical lymph nodes collected in RPMI+ were passed through a 0.1 μ sieve (BD) using additional RPMI+ and centrifuged to separate cells from tissue debris. Retrieved lymph node cells were counted on a hemocytometer and resuspended in PBS to a final concentration of 2 × 10ive mAbs . Followipost-hoc Holm-Sidak's test was used to confirm significant interactions between groups. Results are presented as mean ± SEM. Differences are considered statistically significant when p < 0.05.Statistical analyses were conducted using GraphPad Prism version 7.02 (GraphPad Software Inc.). All data outliers were removed using the ROUT method, and normality of data distribution was determined by visual inspection of histograms. Comparisons between the treatments (short-term and long-term EE) and respective controls were performed using a fixed-effect model of two-way ANOVA. The two-way ANOVA statistical model was chosen to elucidate a two-way interaction effect between the two independent variables (treatment and duration) on the dependent variable . The multiple comparisons post-hoc Holm-Sidak's multiple comparison test. + microglia and GFAP+ astrocytes in the dentate gyrus, CA1, CA2, and CA3 regions of the hippocampus in short- and long-term control and EE groups.Immunopositive microglia and astrocytes were counted in the dentate gyrus, CA1, CA2, and CA3 regions of the hippocampus and analyzed statistically using two-way ANOVA with F = 0.09; p = 0.7615) and the main effects of duration  = 0.15; p = 0.6994) were noted for IBA1+ microglia within the dentate gyrus. However, the main effect of treatment was significant  = 53.1; p < 0.0001). Both short-term and long-term EE treated mice showed significantly higher IBA1+ cell numbers than their respective controls in the dentate gyrus  = 0.09; p = 0.7594), however, the main effects of both duration  = 25.17; p < 0.0001) and treatment  = 23.61; p < 0.0001) were significant. Both short-term and long-term EE treated mice showed significantly higher IBA1+ cell numbers than their respective controls  = 0.14; p = 0.7167), and significant main effects of both duration  = 13.12; p = 0.0017) and treatment  = 5.43; p = 0.0303) were noted for the number of IBA1+ microglia in the hippocampal CA2 region. Both short-term control and EE mice showed significantly higher IBA1+ cell numbers than their respective long-term cohorts  = 0.46; p = 0.5049) but the main effects of both duration  = 42.64; p < 0.0001) and treatment  = 25.30; p < 0.0001) were significant. Both short-term and long-term EE treated mice showed significantly higher IBA1+ cell numbers than their respective controls  = 4.36; p = 0.0497) and the main effect of treatment  = 5.56; p = 0.0286) were noted, but the main effect of duration  =.83; p = 0.3739) was non-significant. On post-hoc analysis, long-term EE mice showed significantly greater number of GFAP+ cells than long-term controls .When we analyzed the number of GFAP+ astrocytes in the hippocampal CA1 region, we noted a non-significant interaction effect  = 0.13; p = 0.7243) and the main effect of treatment  = 0.81; p = 0.3777). The main effect of duration was significant  = 8.02; p = 0.0103). On post-hoc analysis, no significant differences were observed between any of the groups (+ astrocytes than short-term controls at the significance level of p < 0.1 (p = 0.0698).During the analysis of the number of GFAPe groups . HoweverF = 0.07; p = 0.8002), as well as the main effects of both duration  = 0.80; p = 0.3805) and treatment  = 0.11; p = 0.7397) were non-significant for the number of GFAP+ astrocytes in the hippocampal CA2 region  = 2.8; p = 0.1081) and the main effect of duration  = 0.89; p = 0.3573) were non-significant, however, the main effect of treatment was significant  = 6.15; p = 0.0221). Post-hoc analysis revealed significantly higher number of GFAP+ astrocytes in the hippocampal CA3 region of long-term EE mice when compared to long-term controls  and CD8+ (cytotoxic T) cell proportions were measured as an indication of cellular immune response to the duration of the EE treatment. Representative images obtained during the FACS data analysis using FCS Express software are shown in + T cells was found to be non-significant in both short-term and long-term EE groups when compared to respective controls, with p > 0.05 for the interaction effect, as well as for the main effects of treatment and duration  = 11.78; p = 0.0022). The proportion of CD8+ T cells was significantly higher in the long-term EE group when compared with the short-term EE group .The proportions of the T cell early activation marker CD25 on both CD4+ microglia within the dentate gyrus, CA1 and CA3 regions of the hippocampus. Conversely, only the long-term EE enhanced GFAP+ astrocyte number within the dentate gyrus and CA3 regions of the hippocampus when compared to control mice. Evaluation of the T cell population within the cervical lymph nodes revealed that both short-term and long-term EE did not affect the proportion of CD4+ T cells and CD4+ T cell subsets. However, long-term EE mice showed a significantly higher proportion of CD8+ T cells compared to short-term mice, which we observed was due to the significantly higher proportion of CD8+ TN cells in long-term EE mice. Both short-term and long-term EE showed no effect on the proportion of T cell early activation marker CD25.The objective of this study was to determine if the functional effects that we have reported in the recently published study  and CD8+ (cytotoxic T cell), helped us to determine the cellular immune response after short-term and long-term EE treatments. We observed that altered CD8+ (cytotoxic T) but not CD4+ (T helper) cell proportions were associated with the duration of the EE treatment.Ours is the first study to investigate the change in the proportion of T cell subsets in the cervical lymph nodes in response to different durations of EE. The analysis of the total T cell counts, including CD4N cells proliferate and convert into TCM or TEM cells on the recognition of a cognate antigen. TCM cells have little or no effector function but mediate reactive memory—proliferate and differentiate to effector cells in response to antigenic stimulation. TEM cells, which are short-lived, migrate to the site of infection, eliminate the antigenic molecular patterns and undergo apoptosis once the antigens are eliminated. However, some TEM cells also differentiate into memory cells that provide long-term adaptive immunity. Our results suggest that the duration of EE has no differential effects on the proportion of CD4+ T cells and their subsets. However, a longer duration of EE may be beneficial by supporting an increase in the proportion of cytotoxic CD8+ T and CD8+ TN cells as found in middle age when compared with those with short term EE.Mature TN cells also respond by acquiring activated phenotypes and upregulation of early T cell activation markers, such as CD25, which are important in the maintenance of tolerance to self-antigen and prevention of autoimmune disorders . Sex could have significant effects on the reported results and as such we believe an independent study investigating a three-way interaction between treatment, duration of EE and sex would be very useful. Also, although the starting age of long-term EE mice is different from short-term EE mice, it was not possible to have the same starting age. In the latter case, we would have been comparing mice of different ages during behavioral testing (after 4 weeks and 6 months of EE). This would have led to even greater differences not related to the EE since aging is a known risk factor for change in brain functions and underlying molecular factors. The questions we have raised should be explored in future EE studies on rodents, including those in preclinical models of brain disorders.Our EE paradigm differed from the many previous published EE protocols in not having running wheels. We focused on stimulating the environment with toys, tunnels, ladders, and other accessories with extra bedding to stimulate cognitive areas in the absence of wheel-running physical activity. Overall, our results suggest that EE with stimulating cognitive objects for both short-term and long-term increases in the number of immunoreactive microglia, but long-term EE is required to modulate the number of immunoreactive astrocytes, and peripheral T lymphocyte proportion and phenotype during middle age in C57BL/6 mice. Together with the results from our published behavioral study (Singhal et al., The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by University of Adelaide Animal Ethics Committee. Ethics approval number M216-12.Material preparation, data collection, and analysis were performed by GS. JMo assisted GS with the mouse husbandry work and collection of data. FC, CT, and JMa assisted GS with the molecular analysis. AH provided technical guidance during the study as and when needed. FC, MJ, EJ, and BB supervised the project. The first draft of the manuscript was written by GS. All authors commented on previous versions of the manuscript, read and approved the final manuscript and contributed to the study conception and design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accumulating evidence has demonstrated that oxidative stress is associated with depression. Our present study aimed at investigating the antidepressant effect and the possible mechanisms of curcumin (CUR) in chronic unpredictable mild stress- (CUMS-) induced depression model in rats. After exposure to CUMS for four weeks, the rats showed depressive-like behavior, and the depressive-like behaviors in CUMS-treated rats were successfully corrected after administration of CUR. In addition, CUR could effectively decrease protein expression of oxidative stress markers  and increase the activity of CAT. CUR treatment also reversed CUMS-induced inhibition of Nrf2-ARE signaling pathway, along with increasing the mRNA expression of NQO-1 and HO-1. Furthermore, the supplementation of CUR also increased the ratio of pCREB/CREB and synaptic-related protein . In addition, CUR could effectively reverse CUMS-induced reduction of spine density and total dendritic length. In conclusion, the study revealed that CUR relieves depressive-like state through the mitigation of oxidative stress and the activation of Nrf2-ARE signaling pathway. As one of the most common neuropsychiatric illness, depression has affected 300 million people of all ages in the modern world . AccordiIncreasing evidence suggested that oxidative stress is responsible for the development of depression . OxidatiOxidative stress is an etiologic factor in depressive/neurodegenerative disorders that it is often accompanied by deregulation of nuclear factor erythroid-2-related factor 2 (Nrf2) pathway, a key antioxidant mechanism indicated as a promising target for treatment of depression . As a piNumerous evidence has indicated that depression was associated with a range of changes in synaptic form , 21. CycCurcuma longa, which exhibited anti-inflammatory, antioxidant, immunomodulatory, and neuroprotective activities [Curcumin (CUR) is the major active component extracted from tivities , 24. It tivities . Notablytivities , 27. Shetivities . Oxidatitivities . In additivities . HoweverTherefore, in our present study, we aimed to investigate the antidepressant-like effect of CUR in CUMS-induced rats. In addition, to further investigate the possible molecular mechanisms underlying the therapeutic effects of CUR, we also assessed whether the possible antidepressant-like effects of CUR are associated with oxidative stress status and the changes on the activation of Nrf2 in the brain.Male Sprague-Dawley rats (180-220 g) were provided by the Hunan Cancer Hospital Animal Centre. The rats were housed in standard conditions . Except prior to sucrose preference test (SPT), food and water were freely available in the whole experiment. This study was approved by the Animal Care and Use Committee of Hunan Cancer Hospital (protocol number 016/2018). All experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals (Chinese Council).The rats were adapted for one week and the CUMS procedure was performed for four weeks as previously described . 24 h fon = 8): control, CUMS, and CUMS+CUR. CUR  was administrated by oral gavage (100 mg/kg/day) in the CUMS+CUR group, and the rats in control group were treated with the same volume of saline.Rats were randomly divided into three groups . Blood samples and the hippocampus were collected in our study. The whole experimental protocols are shown in The SPT was performed as our previous study . Prior tThe FST was performed as described previously  with minAll the rats were food deprived for 24 h in their home cages before NSFT. A piece of white paper (10 × 10 cm) was placed in an open field (75 × 75 × 40 cm), and a small amount of food was placed on this paper. The rats were allowed to explore the open field for 8 min. The time it took for the rat to approach and take the first bite of the food was defined as the latency time and was recorded in our study. Immediately afterwards, the animals were transferred to their home cage, and the total food intake for the next 5 min was also weighed to avoid the influence of the animals' appetite.The open field apparatus consisted of a 76 × 76 cm gray wooden box with 42 cm high boundary walls. The floor was divided into 25 equal squares by black lines. Each rat was placed in the center of the square and left to explore it freely for 5 min. The number of crossing and rearing was recorded by the observer blind to the treatment condition of the animal. The apparatus was cleaned with ethanol and water to remove olfactory cues.For the determination of serum corticosterone, the blood samples were collected at 13:00-15:00 on day 38 before sacrifice. The collected plasma was centrifuged  at 4°C and stored at -80°C until analysis. The serum corticosterone levels were measured using a commercial ELISA kit  according to the manufacturer's instructions. The standards and samples were all run in duplicates, and the averaged data were used for statistical analysis.β-actin as an internal standard.According to the instruction of manufactory, total RNA was extracted from the hippocampus using TRIzol reagent . The mRNA expression of Nrf2, NQO-1, and HO-1 was determined in our present study. Quantitative PCR was performed on Bio-Rad Cx96 Detection System  using the SYBR green PCR kit  and gene-specific primers. The sequences of gene-specific primers are listed in μl of brain homogenate supernatant and mixed well, and then, the solutions were centrifuged at 1006×g for 10 min. One milliliter of the supernatant was added to 0.5 ml of 0.7% TBA, and then, the mixture was heated for 60 min at 90°C. The absorbance was recorded at 532 nm using UV spectrophotometer. CAT activity was assayed by H2O2 consumption, following Aebi's [Malondialdehyde (MDA) content was determined according to the previous report . Brieflyg Aebi's .μg proteins in each lane. Proteins in the gels were transferred to a polyvinylidene fluoride membrane and blocked for 1 h in 5% nonfat dry milk in TBS-T . The antibodies and concentrations listed below were used overnight at a temperature of 4°C: Nox2 ; 4-HNE ; Nrf2 ; pCREB ; CREB ; BDNF ; PSD-95 ; synaptophysin ; PCNA ; and β-actin . Membranes were then probed with horseradish peroxidase-conjugated secondary antibody for 40 min. After washing, the membranes were dipped in electrochemiluminescence, and immunoblots were analyzed by using the Bioprofl Biolight PC software . β-Actin was used as an internal standard to normalize the signals.For western blot analysis, total protein was prepared from the hippocampus, and the Bradford method was used to determine its concentration. The hippocampus sample was loaded on a precast 12% SDS-PAGE gel with 10 According to the manufacturer's instructions, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) detection kit  was used to assess apoptosis. Apoptotic index was defined as the average percentage of TUNEL-positive cells in 20 nonoverlapping cortical fields under ×200 magnification.Paraffin-embedded tissue sections were rehydrated first in xylene and then in graded ethanol solutions. The slides were then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 h. After incubation with anti-8-OHDG and anti-Nox2 overnight at 4°C, the sections were then washed with PBS and incubated with secondary antibodies. Counter staining was performed using hematoxylin, and the slides were visualized under a light microscope.μm section. The following steps included treatment with ammonia water and acid hardening fixing bath and dehydration with increasing concentrations of alcohol. A digital camera attached with microscopy was used to take images of the tissues for dendritic structure analyzing. Dendritic spine density and total dendritic length analysis were done manually using the Fiji software under ×400 magnification.Golgi staining was performed as previous report . In brieμm thick) were incubated with 5 μM DHE in PBS at 37°C for 30 min in a dark humidified chamber. The sections were then imaged by using the Leica fluorescence microscope .Reactive oxygen species (ROS) was measured by dihydroethidium (DHE) microfluorography as previous study . In briep < 0.05 was considered statistically significant.Statistical Package for Social Science (SPSS) version 18  was used for data analysis in our study. All data were analyzed by one-way analysis of variance (ANOVA) with least significant difference (LSD) post hoc multiple comparisons. All data were presented as means ± SD, and p < 0.01), prolonged immobility time in FST (p < 0.01), and latency time in NSFT (p < 0.01) in comparison with the rats in control group. Our study also observed that the number of crossing (p < 0.05) and rearing (p < 0.01) in OPT was all significantly decreased in the CUMS group. In comparison with the CUMS group, the administration of CUR successfully increased the sucrose preference (p < 0.01), decreased immobility time (p < 0.01) and latency time (p < 0.01), and increased the number of crossing (p < 0.05) and rearing (p < 0.01) in the CUMS+CUR group. In addition, no significant difference of food intake was observed in NSFT.The CUMS group showed reduced source preference in SPT (p < 0.01) in the CUMS group compared with the control group. However, the administration of CUR markedly decreased (p < 0.01) the corticosterone level when compared with the rats in the CUMS group.As displayed in p < 0.01) and 4-HNE (p < 0.01) were significantly increased in the CUMS group as compared to the rats in the control group, and the administration of CUR effectively mitigated CUMS-induced increasing of Nox2 (p < 0.05) and 4-HNE (p < 0.01) was significantly increased in the CUMS group when compared to the control group. The CUR treatment successfully decreased the content of MDA in the CUMS+CUR group when compared to the CUMS group (p < 0.01). In comparison with the rats in the control group, the activity of CAT (p > 0.05) was decreased in CUMS-treated rats but without significance difference. Furthermore, the administration of CUR significantly increased CAT activity in the CUMS+CUR group when compared to the CUMS group (p < 0.05).The immunohistochemical staining results of 8-OHDG and Nox2 are shown in nd 4-HNE p < 0.01;p < 0.05) and nuclear (p < 0.01) all significantly decreased in the CUMS group when compared with the rats in the normal-treated group, and the Nrf2 in nuclear obviously (p < 0.01) increased in the CUMS+CUR group compared to the CUMS group. Interestingly, as shown in p < 0.01) and HO-1 (p < 0.05) all significantly decreased in the CUMS model rats, and CUR treatment significantly prevented the decrease of NQO-1 (p < 0.01) and HO-1 (p < 0.05) in the CUMS+CUR group when compared with the rats in the CUMS group.The Nrf2 levels in cytoplasmic (p < 0.05). Administration of CUR significantly increased pCREB/CREB ratio in the hippocampus compared to the CUS-treated rats (p < 0.01). The protein expression of BDNF (p < 0.01), PSD-95 (p < 0.05), and synaptophysin (p < 0.01) all significantly decreased in the CUMS-treated rats, and CUR successfully reversed the CUMS-induced decrease of these three proteins (p < 0.01). Previous studies have reported that dynamic alterations in synaptic and dendritic structure and function play a pivotal role in the development of depression [p < 0.01) and total dendritic length (p < 0.01) significantly decreased in the dentate gyrus (DG) granule neurons of CUMS-induced rats, and the administration of CUR markedly reversed this effect .As shown in pression , 39. In Our present study demonstrated that the administration of CUR exhibited antidepressant-like activities in CUMS-induced depression model. We investigated the depressive-like behaviors  in rats under CUMS, and chronic administration of CUR normalized behavioral changes in rats exposed to stress. CUR could effectively decrease protein expression of oxidative stress marker . CUR could also activate stress-induced Nrf2-ARE axis inhibition. In addition, long-term treatment with CUR markedly prevented CUMS-induced reduction of BDNF, PSD-95, and synaptophysin expressions. These findings indicate the potential benefits of administration of CUR to reverse the development of depression. Furthermore, the antidepressant mechanism of CUR may be mediated by restoring changes in oxidative stress and the activation of the Nrf2-ARE signaling pathway.The CUMS model has long been used as animal model of depression, and previous study showed that most effects of CUMS could be effectively reversed by antidepressant agents . In our As a stress marker, corticosterone level is widely used for accessing the stress state. More and more evidences showed that the elevated corticosterone level was associated with depressive-like behaviors, and antidepressant-like activity could always induce a reduction of corticosterone level –46. In tThe elevated free radical generation and decreased activity of antioxidants break the balance between oxidant-antioxidant systems, which will always induce oxidative stress . More anAs a transcription factor, Nrf2 is known to play a pivotal role in modulating oxidative stress and exhibiting an important protective role in brain injury and neurodegenerative diseases. As it is known to us, Nrf2 is mainly located in the cytoplasm under physical conditions. However, Nrf2 translocates into the nucleus in response to oxidative stress . In our CREB is involved in the regulation of genes associated with synaptic and neural plasticity . CREB haIn conclusion, our present study suggests that the administration CUR is effective in preventing CUMS-induced depression. Furthermore, the current results suggested that the antidepressant action of CUR may be mediated by restoring changes in oxidative stress, the Nrf2-ARE signaling pathway, and the synaptic and neural plasticity, which might ultimately contribute to its antidepressive-like effect.


Objectives: To study Epstein-Barr virus (EBV) antibody patterns in twin individuals with rheumatoid arthritis (RA) and their healthy co-twins, and to determine the heritability of antibody responses against the EBV encoded EBNA1 protein.Methods: Isotypes of EBNA1 antibodies were measured in 137 RA affected- and 150 healthy twin pairs. We estimated the effect of RA and RA predisposition, anti-citrullinated antibodies (ACPA), IgM rheumatoid factor (RF), the shared epitope (SE) and the PTPN22-T allele (PTPN22) on the level of EBNA1 antibodies. We also determined the heritability of EBNA1 antibody levels.Results: IgA-EBNA1 antibody levels were increased in twins from RA discordant twin pairs irrespective of RA, ACPA or IgM-RF status. The IgG-EBNA1 antibody level was elevated in healthy co-twins from RA discordant twin pairs but not in RA affected twins. The IgM-EBNA1 antibody level was elevated in both RA twins and their healthy co-twins. The effect of RA on the IgA-EBNA1 antibody level was reversed when SE was present and with no effect of PTPN22. The heritability of IgA-, IgG- and IgM-EBNA1 antibody level was 40.6, 65.5, and 54.3%, with no effect of environment shared by the twins.Conclusion: EBNA1 antibody levels are distinctively different between patients with RA and healthy subjects but also between relatives of RA strongly predisposed to RA and healthy subjects. The high level of IgA EBNA1 antibodies associated with RA and a family predisposition to RA is attributable to both genetics incl. the shared epitope and environmental variation. Rheumatoid arthritis is a systemic autoimmune disease which is primarily characterized by peripheral joint synovitis which if left untreated may lead to joint destruction and loss of function. The etiology is unknown, but infections have been proposed as environmental triggers in up to 20% of patients . Viral tA recent meta-analysis on sero-epidemiological associations between EBV and RA did not support the hypothesis that previous infection with EBV predisposes to RA . HoweverTo investigate the association between, RA as well as susceptibility to RA, and EBNA1 antibody isotype levels in RA we studied cohorts of healthy twins and disease discordant twin pairs. In addition, we estimated the heritability of antibody levels and assessed a potential modifying effect of the shared epitope (SE), its QKRAA motif  and the This study was based on two twin cohorts recruited from The Danish Twin Registry . One cohSerum was frozen immediately at −80°C. DNA was isolated from EDTA blood samples and kept at −80 degrees. Zygosity among same-sexed twins was determined by genetic markers.HLA-DRB1 high-resolution genotyping was performed by sequencing using the HLA-DRB1 SBT Resolver kit (Olerup) followed by analysis of data using the Conexio Genomics' Assign SBT software according to the manufacturer's instructions.The following subtypes contain the QKRAA sequence; 0401; 0409; 0413; 0416; 0421; 1419; 1421 (PTPN221 (rs2476601) was determined with end point genotyping using TaqMan SNP Genotyping Assay kit (ThermoFisher Scientific) according to the manufacturer's instruction. PCR was run on a LightCycler 480 II (Roche) starting with an initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 20 s; 62°C for 20 s and 72°C for 20 s.IgM-RF was determined by ELISA using purified IgG as antigen and peroxidase-conjugated F(ab)2 rabbit immunoglobulin  against human IgM as detecting antibody. Detection limit: 1.4 IU/ml, cut off value: 17 IU/ml, clinical sensitivity for RA: 70%, clinical specificity for healthy donors: 95%.Anti-cyclic citrullinated peptide (CCP) IgG antibodies (ACPA) were determined by ELISA  as described by the manufacturer. Clinical sensitivity for RA: 77.4%, clinical specificity for healthy donors: 98.8%. Detection limit 1.6 U/ml, cut off value 25 U/ml.EBNA1 antibody ELISA was determined as previously described  diluted in carbonate buffer  was coated on to NUNC Polysorp microtiter plates  overnight at 4°C for EBNA1 IgG and IgM detection and at room temperature (RT) for IgA detection. After coating, plates were washed 3 × 1 min followed by blocking for 30 min. The samples were subsequently incubated for 1 h at RT in duplicate in both coated and non-coated wells diluted 1:1,000/1:150/1:200 for detection of EBNA1 IgG/IgA/IgM. Following another three washings, the plates were incubated for 1 h at RT with alkaline phosphatase (AP)-conjugated goat antihuman IgG, IgA or IgM . After three more washings, antibodies were quantified using ρ-nitrophenylphosphate (ρ-NPP)  dissolved in AP substrate buffer . After incubations for 30 min for measuring EBNA1 directed IgG antibodies and after 60 min for measuring IgA and IgM antibodies, the absorbances were read on a microplate reader at 405 nm, with background subtraction at 650 nm.Briefly, TTN buffer  was used for blocking, dilutions and washings. Recombinant EBNA1  for included samples. Samples with a calculated titer > 100 U/ml were rerun and normalized relative to the standard.Inter-assay coefficients of variation were 17.9, 7.3, and 14.0% regarding EBNA1-directed IgG, IgA, and IgM, respectively, calculated from the low positive control. Intra-assay coefficients of variation were 9.2, 4.9, and 5.7% regarding EBNA1-directed IgG, IgA, and IgM, respectively, calculated from 16 values derived from the same sample in each assay (a sample known to have an antibody titer in the linear part of the standard curve).Regression models for the three isotypes were adjusted for age, gender and the potential effect of RF, ACPA, and RA. Furthermore, we tested the potential effect of belonging to a twin pair, with at least one twin having RA. We included this effect because we wanted to study if the healthy co-twins from RA discordant twin pairs shared an underlying unobserved effect on the level of EBNA1 antibodies. This was done as healthy co-twins have a relative risk of developing RA of 25 compared to the background population reflecting a high degree of liability to RA in these co-twins , 17.The SE, and its QKRAA subtype, and the PTPN22 C1858T-allele (PTPN22) was measured in pairs with at least one twin being diagnosed with RA. This was done to assess whether the effect of RA on EBNA1 anti-body level was modified by the presence of SE or PTPN22, which are the two major genetic risk factors in RA.The inflammatory condition in RA may lead to high concentrations of immunoglobulins, including those directed against EBV, as well as rheumatoid factor (RF). We have therefore adjusted for the presence of RF in the analysis.Twin data can be used to study the heritability of traits by use of the so called ACDE model, where differences in the covariance structures between DZ and MZ twin pairs contrast the genetic and environmental contributions , 22. TheThe ACDE model for analysis of twin data, is a regression-type model, with a covariance structure that reflects the assumed intra-pair correlations induced by additive (A) and dominant (D) genetic effects and common (C) and individual (E) environmental effects. The E component is merely a term for the measurement error known from conventional regression models. Most data do not allow for estimating both genetic components. A pragmatic approach is therefor to fit both an ACE model and a DCE model and choose the one, which provides the best fit. In addition to estimating the effect of some covariates, the ACDE model also allows for estimating the heritability of a trait defined as the proportion of the total random variation that can be attributed to genetic variation.p-level < 0.05 was chosen as statistically significant.Stata Ver. 14.2 for windows was used for the statistical analyses and a The study was approved by all the regional scientific ethics committees in Denmark (Projekt ID: S-20070088) and the Danish data protection board (J.nr. 2007-41-0747).A total of 283 twin pairs participated in this study, 150 healthy pairs and 133 RA-affected twin pairs .Among the RA pairs 87 were complete (both twins participated) and 46 were incomplete in which only the RA-affected twin participated. Two monozygotic (MZ) and 2 dizygotic same sexed (DZss) pairs, all females, were concordant for RA. Thus, 137 RA affected twins and 83 of their healthy co-twins in addition to 300 twins from healthy pairs participated.The mean age of RA onset was 44 years and the mean age at blood collection was 63.5, 61.7, and 35.2 in RA twins, healthy RA co-twins and healthy twins, respectively .In RA twins 74.5% were females and the sex distribution and the occurrence of erosive or nodular disease were similar across zygosities . The twoRA-affected twin pairs had significantly increased IgA-EBNA1 titers whereas there was no effect by RA itself or any of the covariates RF, ACPA, age or sex. Since there was no difference in IgA-EBNA1 antibody levels between twins with RA and their healthy co-twin we pooled the results . The herThe IgG-EBNA1 titer was significantly higher in healthy co-twins from RA affected twin pairs vs. their RA counterparts and healthy controls. There was no effect of RF, ACPA, age or sex . There wBoth RA-affected twins and their healthy co-twins from RA-affected twin pairs had significantly elevated IgM-EBNA1 titer compared to healthy twin pairs. Belonging to an RA-affected twin pair fitted marginally, but insignificantly better than a model including RA-status. Male sex and increasing age were associated with a lower IgM-EBNA1 titer . The herp < 0.01) in SE negative RA and 0.3175  in SE positive RA and −0.3459  in QKRAA negative RA and 0.5126  in QKRAA positive RA. Inclusion of the interaction terms resulted in significantly better model fits. Thus, we observed a crossover interaction with reversal of the RA effect when SE and QKRAA was present. That is, the regression coefficient of RA on IgA-EBNA1 antibodies was negative in SE negative RA and QKRAA negative RA and positive in SE positive and QKRAA positive RA. Hence, the effect of RA on IgA-EBNA1 level goes in opposite directions in SE (QKRAA) positive RA compared to SE (QKRAA) negative RA. In other words, the presence or absence of the shared epitope determines whether the effect of both RA per se and RA disposition, increases or decreases the level of IgA-EBNA1 antibodies.We found significant effect modifications of SE and the QKRAA subtype on the association between RA and the level of IgA-EBNA1 antibodies but not the other isotypes. The regression coefficient of RA was −0.8844  and the Danish Data Protection board (J. nr. 2007-41-0747). We obtained informed written consent from all participants in the study. The patients/participants provided their written informed consent to participate in this study.AS conceived the study. AS, RH, PJ, and GH analysis and interpretation of the data and prepared the manuscript. GH, MW, AD, KK, and MJ contributed reagents/materials/analysis tools. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. NAFLD affects approximately one-quarter of the world’s adult population, and its incidence is rapidly increasing . The NAFTherapeutic options for NASH patients are still limited and are therefore urgently needed. Weight loss, healthy food choices and physical activity are the only established therapeutic strategies established thus far, as they also attenuate the underlying metabolic dysfunction . MoreoveA novel class of bioactive lipids called branched fatty acid esters of hydroxyl fatty acids (FAHFAs) was recently discovered . FAHFA fIn the present study, we show two important results. First, we show alternative ways to implement the 3R principle in NASH research by establishing a new complementary model of in vitro steatotic hepatocytes. Second, our research led to the development of a nondrug therapy based on dietary supplements or nutritional modifications. We showed the therapeutic effect of 9-PAHSA against lipotoxicity on steatotic hepatocytes and a preventive effect on HepG2 cells. The outcome of 9-PAHSA administration was both preventative and therapeutically effective in hepatocytes with limited damage.Endogenous 9-PAHSA has been shown to have a role in insulin sensitivity and as an anti-inflammatory and antioxidant agent ,11. In tTherefore, HepG2 cells were incubated with 9-PAHSA for 12 h and then exposed to oleic acid (OA) for 6 h to induce steatosis. Bovine serum albumin (BSA) was used as a control B. ProperWe demonstrated that 9-PAHSA had a protective effect on mitochondrial respiration following OA incubation. Thus, we wondered whether 9-PAHSA could be used as a therapeutic agent for steatosis. To evaluate this possibility, we isolated hepatocytes from 8-week-old C57BL/6 mice and used as before HepG2 cells. The hepatic cells were incubated with three different concentrations of OA for 24 h to induce steatosis A.The WST-1 cell proliferation assay showed a significant concentration-dependent reduction in the viability of the OA-treated primary murine hepatocytes (PMH) and HepG2 cells. Exposure to DMSO as a diluent agent for 9-PAHSA did not alter the viability of the cells B,C. SubsThe effect that the viability of PMH and HepG2 with steatosis could be improved by the administration of 9-PAHSA is remarkable. However, the mechanisms behind remain still unclear. Among other things, our suggestion would be a direct effect on the lipid accumulation in the cells. Therefore, we introduced here a method to quantify the amount of Oil Red O incorporated by OA-treated hepatic cells.To prove the steatotic induction in PMH and HepG2 cells, we used Oil Red O staining. Steatotic cells exhibited a strong staining showing the intracellular lipid accumulation A,B AdditIn this paper, we reported two new in vitro models for the complementary study of NASH by inducing hepatic steatosis with OA, which is a fat-carrying agent. In addition, we tested, for the first time, 9-PAHSA in an experimental NASH model. These in vitro methods support the 3R principle used in animal experimentation: an alternative to animal experiments (replace), use of the absolute minimum number of animals (reduce) and ensure minimal animal stress (refine). With these models, we showed the positive effects of 9-PAHSA used for both prevention and treatment in NASH-like PMH and HepG2 cells, which can be used as substitutes for primary hepatocytes. Additional research in other NASH diet models would complement our results and investigation.The role of 9-PAHSA as an antidiabetic and anti-inflammatory agent is controversial. Increased and decreased levels of endogenous PAHSAs in serum have been found in insulin-resistant patients and insulin-sensitive mice, respectively ,11. PAHSNevertheless, Pflimlin et al. using a murine model of diet-induced obesity reported no detection of antidiabetic effects after PAHSA treatment . These cFAHFAs are bioactive lipids present not only in mammalian tissues but also in a diverse range of foods. Clementine, whole grain oat, garlic, pineapple and avocado are examples of food with the highest number of FAHFA families. The most studied group of FAHFAs, PAHSAs, are mostly found in carrot, mango, banana, whole grain oat and apple . In partUsing PMH, we showed that OA could be used as a fat-carrying agent to mimic NASH in vitro. The viability of the NASH-like cells was dramatically reduced in an OA-concentration-dependent manner. The capacity of OA in inducing steatosis was previously shown in rat primary hepatocytes . AlthougAfter the induction of steatosis with OA, treatment with 9-PAHSA led to increased cell viability that was concentration dependent. While low 9-PAHSA concentrations seemed to have no effect on cell toxicity, high concentrations increased it. To our knowledge, no studies have dealt with PAHSA overdose. This bioactive lipid is produced endogenously in mammals and exists in food in a small amount. This suggests that the risk of toxicity due to ingested food is low. However, it has been established that even most essential vitamins and minerals have negative health effects when overdosed .A recent publication reported that 9-PAHSA treatment increases islet β cell viability during cytokine-induced cell death. This outcome was due to PAHSAs attenuating ER stress , and thiTo elucidate the 9-PAHSA impact on hepatosteatosis and NASH, additional investigation is required. Utilizing human hepatocytes and generating organoids would be advantageous in these studies.The growing epidemic of obesity and fatty liver requires new strategies for prevention and treatment. Our data revealed that 9-PAHSA could be used in steatotic liver. The inclusion of 9-PAHSA-containing food in the diet may help in the prevention of NASH in patients at risk. Considering the hypoglycemic and anti-inflammatory roles of 9-PAHSA, its incorporation into the diet can additionally benefit chronic diseases such as obesity, metabolic syndrome and diabetes. Since 9-PAHSA treatment cannot rescue severely damaged cells, an alternative is to develop synthetic products and incorporate them into practical diet formulations as supplemental aids in the treatment of NASH.C57Bl/6J mice were maintained under specific pathogen-free conditions at the Central Animal Facility of University Hospital Essen .Animal care and experiments were performed in accordance with institutional and national guidelines. All animal experiments were performed according to protocols approved by the Animal Welfare Commission of University Hospital Essen  and the local Ethics Animal Review Board . The procedure for mouse hepatocyte preparation has been adapted to the use dead animals sacrificed by cervical dislocation. Under LANUV’s regulations, no additional applications are required to perform that technique. C57Bl/6J mice by collagenase perfusion as described previously [PMH were prepared from eight-week-old eviously .v/v) was replaced with fatty acid-free BSA (1% v/v). The medium for culturing the PMH was replaced with fresh medium containing 250 μM, 500 μM, or 1000 μM OA and incubated with cells for 24 h. For HepG2 cells, the medium was replaced by 500 μM oleic acid for six hours after 9-PAHSA treatment.Oleic acid  stock solution was complexed to BSA at a 5:1 molar ratio. For exposure to OA, culture medium supplemented with fetal bovine serum  for 20 min. Cells were rinsed until no excess stain was seen. Cells were incubated with hematoxylin for 1 min followed by rinsing steps. They were covered with distilled water and visualized under a microscope. For dye elution, an incubation with 100% 2-propanol for 10 min was performed. Absorbance was measured at 492 nm with a spectrophotometer.For HepG2 cells, intracellular lipid accumulation was additionally visualized after 6 h 9-PAHSA treatment and measured by absorbance at 492 nm.9-PAHSA  was dissolved in 100% DMSO to a final stock concentration of 100 mM. For the PMH, the medium for steatosis induction was replaced with fresh medium containing 5 μM, 10 μM, 20 μM or 40 μM 9-PAHSA for six hours. For the prevention experiments with HepG2 cells, medium containing 9-PAHSA was added to the cells, which were cultured for 12 h before steatosis induction.4 cells/well. After steatosis induction and 9-PAHSA treatment, cell viability was measured using a cell proliferation reagent WST-1 kit  according to the manufacturer’s instructions. The absorbance was measured by a spectrophotometer .PMH were cultured overnight in 96-well plates at a density of 5 × 104 cells/100 μL medium/well. After pretreatment with 9-PAHSA, the culture medium was replaced with fresh medium containing OA.HepG2 cells were plated overnight on collagen-coated, XF 24-well cell culture microplate at a density of 4 × 10https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/seahorse-wave-desktop-software-740897) was used. Data are expressed as mean ± SEM from 3 technical replicates.Mitochondrial OCR was assessed with a Seahorse XF24 Analyzer. The measurements were performed according to the manufacturer’s instructions. The following concentrations were used: 1 μM oligomycin, 0.25 μM FCCP and a mix of 0.5 μM rotenone and 0.5 μM antimycin A. Agilent Seahorse Wave Desktop software, Wave 2.6.1, for the assay design and data analysis . Data are presented as the mean ± standard error of the mean.Prior analysis, WST-1 and Oil Red O data were normalized to their corresponding controls. The unpaired Student’s two-tailed